Integrins are a family of .alpha..beta. heterodimers that mediate adhesion of cells to extracellular matrix proteins and to other cells (Clark et al., Science (1995) 268: 233-239). Integrins also participate in signal transduction, as evidenced by either an alteration in adhesive affinity of cell surface integrins in response to cellular activation (termed inside-out signal transduction) or by affecting intracellular signaling pathways following integrin-mediated adhesion (termed outside-in signal transduction). Many biological responses are dependent at least to some extent upon integrin-mediated adhesion and cell migration, including embryonic development, hemostasis, clot retraction, mitosis, angiogenesis, cell migration, inflammation, immune response, leukocyte homing and activation, phagocytosis, bone resorption, tumor growth and metastasis, atherosclerosis, restenosis, wound healing, viral infectivity, amyloid toxicity, programmed cell death and the response of cells to mechanical stress.
The integrin family consists of 15 related known .alpha. subunits (.alpha.1, .alpha.2, .alpha.3, .alpha.4, .alpha.5, .alpha.6, .alpha.7, .alpha.8, .alpha.9, .alpha.E, .alpha.V, .alpha.IIb, .alpha.L, .alpha.M, and .alpha.X) and 8 related known .beta. subunits (.beta.1, .beta.2, .beta.3, .beta.4, .beta.5, .beta.6, .beta.7, and .beta.8, Luscinskas et al., FASEB J (1994) 8:929-938). Integrin .alpha. and .beta. subunits are known to exist in a variety of pairings as indicated in FIG. 1. Integrin ligand specificity is determined by the specific pairing of the .alpha. and .beta. subunits, although some redundancy exists as several of the integrins are known to bind the same ligand. FIG. 2 shows the sequences of the cytoplasmic domains of GPIIb and GPIIIa, including the cytoplasmic domains of other .alpha. and .beta. subunits, respectively, that have homologous cytoplasmic domains. Most integrins containing the .beta.1, .beta.2, .beta.3, .beta.5, .beta.6, and .beta.7 subunits have been found to transduce signals (reviewed by Hynes, Cell (1992) 62:11-25). Integrins are involved in both "inside-out" and "outside-in" signaling events.
Various pathologies associated with integrin-related defects are known. For example, inherited deficiencies of GPIIb-IIIa (also termed .alpha.II.beta.3) content or function have been described (termed Glanzmann's thrombasthenia) and are characterized by platelets that do not bind adhesive proteins and therefore fail to aggregate, resulting in a life-long bleeding diathesis. Inhibitors of the binding of fibrinogen and von Willebrand factor to GPIIb-IIIa have been described and have been found to block platelet aggregation in vitro and to inhibit clinical thrombosis in vivo (The EPIC Investigators, New England Journal of Med (1994) 330:956-961; Tcheng, J. E. et al., Circulation (1995) 91:2151-2157). Also, leukocyte adhesion deficiency (LAD) results from the absence of a .beta.2 subunit.
A. Inside-Out Signaling
Inside-out signal transduction has been observed for .beta.1, .beta.2, and .beta.3 integrins. (Hynes, R. O. Cell (1992) 69:11-25; Phillips, D. R. et al., Cell (1991) 65:359-362; Smyth, S. S. et al., Blood (1993) 81:2827-2843; Ginsberg, M. H. et al., Thromb Haemostasis (1993) 70:87-93; Juliano, R. L. et al., Cell Biol (1993) 120:577-585; Rouslahti, E., J Clin Invest (1991) 87:1-5.
Perhaps the most widely studied integrin that is involved in inside-out signaling is GPIIb-IIIa, the receptor for the four adhesive proteins, fibrinogen, von Willebrand factor, vitronectin and fibronectin, on stimulated platelets (Phillips, D. R. et al., Blood (1988) 71:83143). The binding of adhesive proteins to GPIIb-IIIa is required for platelet aggregation and normal hemostasis and is also responsible for occlusive thrombosis in high shear arteries.
GPIIb-IIIa is known to be involved in inside-out signal transduction because GPIIb-IIIa on the surface of unstimulated platelets is capable of recognizing only immobilized fibrinogen. In response to platelet stimulation by agents such as thrombin, collagen and ADP, GPIIb-IIIa becomes a receptor for the four adhesive proteins in the previous paragraph, and the binding of fibrinogen and von Willebrand factor causes platelets to aggregate. A monoclonal antibody has been described which detects the activated, receptor competent state of GPIIb-IIIa, suggesting that the conformation of the receptor competent form of GPIIb-IIIa differs from that of GPIIb-IIIa which does not bind soluble fibrinogen or von Willebrand factor (Shattil, S. J. et al., J Biol Chem (1985) 260:11107-11114). It has been postulated that inside-out GPIIb-IIIa signal transduction is dependent on cellular proteins that act to repress or stimulate GPIIb-IIIa activation (Ginsberg, M. H. et al., Curr Opin Cell Biol (1992) 4:766-771).
.beta.2 integrins on leukocytes also respond to inside-out signal transduction which accounts, for example, for the increased binding activity of LFA-1 on stimulated lymphocytes and the increased binding activity of MAC-1 on stimulated neutrophils (reviewed by Springer, T., Curr Biol (1994) 4:506-517).
B. Outside-In Signaling
Most integrins can be involved in outside-in signal transduction as evidenced by observations showing that binding of adhesive proteins or antibodies to integrins affects the activities of many cells, for example cellular differentiation, various markers of cell activation, gene expression, and cell proliferation (Hynes, R. O. Cell (1992) 69:11-25). The involvement of GPIIb-IIIa in outside-in signaling is apparent because the binding of unstimulated platelets to immobilized fibrinogen, a process mediated by GPIIb-IIIa, leads to platelet activation and platelet spreading (Kieffer, N. et al., J Cell Biol (1991) 113:451-461).
Outside-in signaling through GPIIb-IIIa also occurs during platelet aggregation. Signaling occurs because fibrinogen or von Willebrand factor bound to the activated form of GPIIb-IIIa on the surface of stimulated platelets, coupled with the formation of platelet-platelet contacts, causes further platelet stimulation through GPIIb-IIIa signal transduction. In this manner, binding of adhesive proteins to GPIIb-IIIa can both initiate platelet stimulation or can augment stimulation induced by the other platelet agonists such as ADP, thrombin and collagen. The binding of soluble fibrinogen to GPIIb-IIIa on unstimulated platelets can also be induced by selected GPIIb-IIIa antibodies such as LIBS6 (Huang, M -M. et al., J Cell Biol (1993) 122:473-483): although platelets with fibrinogen bound in this manner are not believed to be stimulated, such platelets will aggregate if agitated and will become stimulated following aggregation through GPIIb-IIIa signal transduction.
Outside-in integrin signal transduction results in the activation of one or more cascades within cells. For GPIIb-IIIa, effects caused by integrin ligation include enhanced actin polymerization, increased Na.sup.+ /H.sup.+ exchange, activation of phospholipases, increased phosphatidyl turnover, increased cytoplasmic Ca.sup.++, and activation of kinases. Kinases known to be activated include PKC, myosin light chain kinase, src, syk and pp125FAK. Kinase substrates identified include pleckstrin, myosin light chain, src, syk, pp125FAK, and numerous proteins yet to be identified (reviewed in Clark, E. A. et al., Science (1995) 268:233-239). Many of these signaling events, including phosphorylations, also occur in response to ligation of other integrins (reviewed in Hynes, R. O. Cell (1992) 69:11-25). Although these other integrins have distinct sequences and distinct .alpha.-.beta.3 parings that allow for ligand specificity, the highly conserved nature of the relatively small cytoplasmic domains, both between species and between subunits, predicts that related mechanisms will be responsible for the transduction mechanisms of many integrins.
C. Signal Transduction
Despite the numerous observations on the binding of cytoplasmic proteins to GPIIb-IIIa and other integrins, it is not yet known whether the binding of any of these is involved in integrin signal transduction nor is it known what regulates their binding to the integrin. There have been several attempts to determine whether phosphorylation of the cytoplasmic domain of GPIIIa and .beta.1 is responsible for GPIIb-IIIa signal transduction. These studies, discussed below, apparently originated from the suggestion made following the determination of the primary sequence of GPIIIa and .beta.1 which showed that tyrosine 747 on the cytoplasmic domain of GPIIIa (and tyrosine 788 on the cytoplasmic domain of .beta.1) was a possible phosphorylation site as it existed within a motif similar to a tyrosine which exists in the cytoplasmic domains of the epidermal growth factor and insulin receptors which are known phosphorylation sites (Fitzgerald, L. et al., J Biol Chem (1987) 262:3936-3939; Tamkun, J. W. et al., Cell (1986) 46:271-282).
Nonetheless, the involvement of the cytoplasmic domain of GPIIb-IIIa in integrin signal transduction is inferred from mutagenesis experiments. Deletion of the cytoplasmic domain of GPIIb results in a constitutively active receptor that binds fibrinogen with an affinity equivalent to the wild-type complex, implying that the cytoplasmic tail of GPIIb has a regulatory role (O'Toole, T. E. et al., Cell Regul (1990) 1:883-893). Point mutations, deletions and other truncations of GPIIb-IIIa affects the ligand binding activity of GPIIb-IIIa and its signaling response (Hughes, P. E. et al., J Biol Chem (1995) 270:12411-12417; Ylanne, J. et al., J Biol Chem (1995) 270:9550-9557).
Chimeric, transmembrane proteins containing the cytoplasmic domain of GPIIIa, but not of GPIIb, inhibit the function of GPIIb-IIIa (Chen, Y. -P. et al., J Cell Biol (1994) 269:18307-18310), implying that free GPIIIa cytoplasmic domains bind proteins within cells and that this binding is necessary for normal GPIIb-IIIa function. Several proteins have been shown to bind either the transmembrane domains or the cytoplasmic domains of GPIIb or GPIIIa.
CD 9, a member of the tetraspanin family of proteins (Lanza, F. et al., J Biol Chem (1991) 266:10638-10645), has been found to interact with GPIIb-IIIa on aggregated platelets. .beta.3-endonexin, a protein identified through two hybrid screening using the cytoplasmic domain of GPIIIa as the "bait", has been found to interact directly and selectively with the cytoplasmic tail of GPIIIa (Shattil, S. et al., Throm and Haemost (1995) 73:1190). .beta.3-endonexin shows decreased binding to the GPIIIa cytoplasmic domain containing the thrombasthenic S752-P mutation. It is not yet known whether either of these GPIIIa-binding proteins are involved in signal transduction.
Cytoplasmic proteins that bind to .alpha.V.beta.3 have also been described which may be interacting with the integrin at the GPIIIa cytoplasmic domain sequence. Bartfeld and coworkers (Bartfeld, N. S. et al., J Biol Chem (1993) 268:17270-17276) used immunoprecipitation from detergent lysates to show that a MW=190 kDa protein associates with the .alpha.V.beta.3 integrin from PDGF-stimulated 3T3 cells. IRS-1 was found to bind to the .alpha.V.beta.3 integrin following insulin stimulation of Rat-1 cells stably transfected with DNA encoding the human insulin receptor (Vuori, K. et al., Science (1994) 266:1576-1578).
.beta.1-containing hybrid proteins also have a dominant negative effect on integrin function implying that .beta.1 integrins also bind cytoplasmic proteins (LaFlamme, S. E. et al., J Cell Biol (1994) 126:1287-1298). The importance of the cytoplasmic domain of .beta.1 is underscored by the demonstration that its removal markedly reduces the adhesive activity of the integrin .alpha.5.beta.1 (Hyashi, Y. et al., J Cell Biol (1990) 110:175-184; Ylanne, J., et al., J Cell Biol (1993) 122:223-233). Mutations of defined sequences of the cytoplasmic domain of .beta.1 have been shown to decrease integrin recruitment to adhesion plaques (Reszka, A. A. J Cell Biol (1992) 117:1321-1330). Several 11 cytoplasmic domain binding proteins have been described. Otey and coworkers (Otey, C. A. et al., J Biol Chem (1993) 268:21193-21197) have used synthetic peptides to map the binding site for .alpha.-actinin within the cytoplasmic domain of .beta.1. Talin binding to a peptide corresponding to the cytoplasmic domain of .beta.1 has been observed (Horwitz, A., et al., Nature (1986) 320:531-533). Argraves and coworkers (Argraves, W. S. et al., Cell (1989) 58:623-629) also used synthetic peptides to show that fibulin bound to the cytoplasmic domain of .beta.1. The NH.sub.2 -terminal, noncatalytic domain of pp125FAK has been found to directly bind to the cytoplasmic tail of .beta.1 and to recognize integrin sequences distinct from those involved in binding to .alpha.-actinin (Schaller, M. D. et al., J Cell Biol (1995) 130:1181-1187). Integrin-associated kinase (IAK) is a tyrosine kinase that has been found to bind to the cytoplasmic tail of .beta.1 (Hannigan, G. E. et al., Nature (1996) 379:91-96).
In order to determine whether or not GPIIIa was phosphorylated on tyrosine residues as a consequence of platelet activation, the following experiments were performed. GPIIb-IIIa from control and thrombin-stimulated platelets was analyzed for changes in phosphorylation and it was observed that stimulation caused an increase in the phosphorylation of GPIIIa, but that the phosphorylation was primarily on serine, with no detectable phosphorylation of tyrosine (Parise, L. V. et al., Blood (1990) 75:2363-2368). Consistent with these findings, a variant of Glanzmann's thrombasthenia has been described where the deficiency of the platelet aggregation response has been attributed to the replacement of a serine residue in the cytoplasmic tail of GPIIIa by a proline residue (Chen, Y. -P. et al., Proc Natl Acad Sci USA (1992) 89:10169-10173). This implies that the sequence that occurs for normal GPIIIa is required for GPIIb-IIIa signal transduction, possibly involving the activation of the receptor function.
Other studies have shown that the sequences of the cytoplasmic domains of GPIIIa, .beta.1 and .beta.2 which contain tyrosines are important for normal functioning of GPIIb-IIIa and of other integrins. Substitution of tyrosine 747 by alanine in GPIIIa transfected into CHO cells abolished GPIIIa-mediated cell spreading, blocked the recruitment of GPIIb-IIIa to preestablished adhesion plaques, and decreased the ability of GPIIb-IIIa to mediate internalization of fibrinogen-coated particles (Ylanne, J. et al., J Biol Chem (1995) 270:9550-9557). Additional experiments in this study showed further that substitution of tyrosine 759 by alanine decreased cell spreading and the recruitment of GPIIb-IIIa to plaques, while deletion of the carboxy terminal pentapeptide that contains this sequence had an even more pronounced effect on the function of the integrin. These authors concluded integrin-mediated cell spreading does not occur because the factors that are absolutely required for integrin-mediated cell spreading cannot bind either the GPIIIa truncated at residue 757 or to the integrin with tyrosine 747 on GPIIIa substituted by alanine. Point mutations in homologous domains in .beta.1- and .beta.2-containing integrins also suggest that these domains are functional as these mutations affect integrin-cytoskeletal interactions (Reszka, A. A. et al., J Cell Biol (1992) 117:1321-1330) and integrin activation (Hibbs, M. L. et al., J Exp Med (1991) 174:1227-1238), respectively. Similarly, an NPXY SEQ ID NO: 27 motif in the integrin .beta.3 cytoplasmic subunit tail appears necessary for melanoma cell migration (Filardo, E. J. et al., J Cell Biol (1995) 130:441-450; O'Toole, T. E. J Biol Chem (1995) 270:8553-8558).
Some commentators have suggested that the phosphorylation of isolated cytoplasmic tyrosine residues was implicated in signal transduction. For example, GPIIb and IIIa isolated from human platelets were reported to serve as substrates for PP.sub.60.sup.c-src (Findik, D. et al, FEBS (1990) 262:1-4). The tyrosine(s) on the cytoplasmic domain of GPIIIa also have been found to be an in vitro substrate for src (Elmore, M. A. et al., FEBS (1990) 269:283-287), but it has not been demonstrated that src phosphorylates GPIIIa in vivo.
Others have reported that these tyrosine residues are not phosphorylated during normal integrin function (Hillery, C. A. et al., J Biol Chem (1991) 266:14663-14669). Thus, neither of the GPIIIa containing integrins, GPIIb-IIIa or .alpha.V.beta.3, are believed to be phosphorylated on tyrosine.
Tyrosine phosphorylation of .beta.1 has been observed, however, but only in cells overexpressing vSrc (Hirst, R. et al., PNAS USA (1986) 83:6470-6474). .beta.1 phosphorylation coincides with a decrease in the ability of the .alpha.5.beta.1 integrin to mediate cell adhesion (Horwitz, A. et al., Nature (1986) 320:531-533) and a decrease in the ability of this integrin to localize to focal adhesion plaques (Johansson, M. W. et al., J Cell Biol (1994) 126:1299-1309). Increased phosphorylation of the cytoplasmic domain of .beta.1 may decrease the binding of talin (Tapley, P. et al., Oncogene (1989) 4:325-333). The available data on .beta.1 integrins suggest that tyrosine phosphorylation of .beta.1 has a negative effect on its function and that tyrosine phosphorylation of .beta.1 may be associated with a transformation phenotype. Similarly, phosphorylation of two conserved tyrosines in the cytoplasmic domain of the integrin .beta..sub.ps subunit was found to be unnecessary for developmental functions in Drosophila (Grinblat, Y. et al., Development (1994) 120:91-102).
Integrin binding to adhesive proteins and integrin signal transduction have a wide variety of physiological roles, as identified above. Enhanced signaling through integrins allows for increased cell adhesion and activation of intracellular signaling molecules which causes enhanced cell mobility and growth, enhanced cell responsiveness, and modulations in morphological transformations. Although integrins responsible for cellular function have been described and signaling events are beginning to be elucidated, the mechanism by which integrins transduce signals remains to be determined. Identification of the event(s) which allow for integrin interactions with cytoplasmic signaling molecules will greatly enhance the understanding of integrin function and will provide for agents which can modulate integrin function. Such agents will be useful for the treatment and diagnosis of a wide spectrum of pathologies, including the processes described above. The present invention describes the event which allows for the interaction in vivo of GPIIb-IIIa with intracellular signaling molecules and peptide structures which can be used to modulate these signaling events.